Electrophoretic analysis is a powerful and widely used technique in the fields of biochemistry and molecular biology. The advent of recombinant DNA technology, the rapid growth of the polymerase chain reaction technology, and the initiative to sequence the human genome have further stimulated its use and development, particularly for the separation of nucleic acids. Nucleic acid analyses are carried out on sample mixtures which range in size from oligonucleotides several nucleotides in length to very large DNA fragments millions of base pairs in length. Agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) are the two main types of electrophoresis used for analysis of nucleic acids. In most cases the short to intermediate sized nucleic acid fragments [10 to 1000 base pairs (bp)] are separated in polyacrylamide slab gels arranged in vertical formats, and the intermediate to high molecular weight nucleic acid fragments (100 to 100,000 nucleotides) are separated in agarose slab gels arranged in a horizontal formats. A special technique called pulse field electrophoresis is used to resolve very large DNA fragments up to 5 megabases. In general, slab gels used in these procedures range from so-called mini-gels which are approximately 5 cm.times.5 cm to the more standard sized gels which are 20 cm.times.20 cm formats. These formats provide running distances of 5 to 20 centimeters in which the separations occur.
In the case of DNA sequencing, where resolution of DNA fragments varying by one nucleotide is required, polyacrylamide gels from 20 cm to 40 cm are commonly used, and in some cases gels as long as 100 cm have been used. Improvements in DNA sequencing using polyacrylamide slab gels has been achieved mainly by using thinner and lower percentage polyacrylamide gels. Presently, using the most optimal techniques and equipment (Applied Biosystems, Inc., Automated Fluorescent DNA Sequencer) a 300-400 base sequence determination on slab gel with a 25 cm running distance takes four to six hours to complete. More recently, a newer technique called capillary electrophoresis has been developed for DNA sequencing applications. This technique uses very narrow diameter capillary tubes (50 .mu.m to 100 .mu.m) containing low percentage polyacrylamide gels. Separations comparable to the slab gel formats can be carried out in 30 to 40 minutes. The improved speed with the capillary format comes primarily from being able to apply higher voltages to the very thin, low percentage polyacrylamide gel; however, gel lengths of 40 to 70 centimeters are still required to achieve the resolution. Thus, in the almost twenty years of development in electrophoretic techniques, the linear dimension or length of gel required for the high resolution separation of nucleic acid fragments has stayed approximately the same.
A few early attempts were made to investigate the potential for microelectrophoresis. Edstrom, (Biochem. Biophys. Acta, 22:378, 1956) first described a microtechnique for the electrophoretic separation of purine and pyrimidine bases along a silk thread. Matioli et al., (Science, 150:1824, 1965) have separated hemoglobin variants on polyacrylamide fibers. In this work, 20% polyacrylamide gels were used to separate hemoglobin variants from single cells. Hemoglobin molecules (MW .sup..about. 64,000) with molecular radii of 2.66 nm are not pore size limited in 20% polyacrylamide gels, thus the separation occurs by the "normal gel sieving process". See Andrews, A. T. in "Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications", Oxford University Press, N.Y., chapter 2, pp. 5-74 (1986). As will be shown, the present invention is concerned with the microelectrophoretic separation of molecules whose molecular radius, stokes radius, or radius of gyrations are significantly greater than the gel pore size.
Other separation systems have been described that were referred to as mini-gel electrophoresis, that emphasize separation of proteins. Grossbach, U. in "Electrophoresis and Isoelectrofocusing in Polyacrylamide Gels", eds. Allen et al, Walter de Gruyter, N.Y., p.207 (1974), describes separation of proteins in 50 .mu.m to 100 .mu.m diameter capillaries tubes which were 2.5 centimeters long, using standard gels and buffers. Neuhoff et al., Biochem J., 117:623 (1970) and Bispink et al, in "Electrofocusing and Isotachophoresis", eds. Radola et al, Walter de Gruyter, N.Y., p125 (1977) describe analytical PAGE on proteins carried out using 5 centimeter capillary tubes. "Micro versions" of polyacrylamide slab gels have been prepared on 75 mm.times.25 mm microscope slides [Maurer et al, Anal. Biochem., 46:19 (1972)] and on 82 mm.times.102 mm glass microscope slides [Matsudaira et al, Anal. Biochem., 87:386 (1978)]. In all of the above work, gel lengths or running distances are at least five to forty times longer than described by the present invention; the above described systems are more accurately described as mini-gels or scaled down version of large gels. In the above work, standard gel formulations were used. That is, gel formulations in the above systems do not significantly deviate from what is used for the corresponding large scale separation. More importantly, in the above systems the molecules (proteins) are being separated by the "normal sieving process" which occurs for molecules which have a molecular radius that is smaller than the gel pore size of the separation medium. Finally, the term "micro" refers more accurately to the width, diameter, or thickness of the gels, rather than to the length or linear dimension.
Gradient gel electrophoresis is a technique in which a gel matrix having an increasing concentration of polyacrylamide (3% to 40%) along the separation axis is used to separate macromolecules in a wide range of sizes. In gradient gel electrophoresis the rate of migration of the components through a gel gradient varies inversely with time. After a sufficient electrophoresis time a stable pattern develops in which the different components continue to move slowly but their relative positions remain constant. That is, as the components reach the gel pore size that is close to their own size (molecular radius), their terminal velocity approaches zero. See, for example, Andrews, A. T., in "Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications", Oxford University Press, N.Y., chapter 4, pp. 93-116 (1986). A great deal of theoretical work and application of techniques for determining molecular weights and molecular radii of proteins has been described by Rodbard et al, Anal. Biochem., 40:95-134 (1971); Manwell, Biochem. J., 165:487-495 (1977); and Campbell et al, Anal. Biochem., 129:31-36 (1983).
DNA separation by using gradient gel electrophoresis has been described by Jeppesen, Anal. Biochem., 58:195-207 (1974). DNA fragments of molecular weights from about 7.times.10.sup.4 daltons (114 bp) to about 14.times.10.sup.6 daltons (21,226 bp) were separated on linear gradient polyacrylamide gels having concentrations from 3.5% to 7.5% or from 2.5% to 7.5% with a crosslinker (C) concentration ranging from 2.5% to 5%. The gels described by Jeppeson were 14 cm long.times.14 cm wide.times.0.3 cm in thickness. Electrophoresis was carried out at 10 volts/cm from 16 to 20 hours, until the DNA fragments reached terminal velocities that approached zero. According to Jeppesen, maximum separation is achieved as the fragments approach zero velocity (gel pore limit), and further increase in running time results in little change in band position. Table 1 below shows the approximate polyacrylamide concentration (%T) or gel pore limit at which point the separated DNA fragments reached terminal velocities:
TABLE 1 ______________________________________ DNA Fragment % Gel (% T) Terminal Fragment MW (daltons) Size (bp).sup.1 Velocity Reached ______________________________________ Lambda DNA/R1 (1) 13.7 .times. 10.sup.6 21,226 3.8 (2) 4.5 .times. 10.sup.6 7,421 3.9 (3) 3.5 .times. 10.sup.6 5,804 3.9 (4) 3.0 .times. 10.sup.6 5,643 3.9 (5) 3.5 .times. 10.sup.6 4,878 4.0 (6) 2.3 .times. 10.sup.6 3,530 4.2 SV40 DNA/R A 6.5 .times. 10.sup.5 .about.1,000 5.2 B 4.2 .times. 10.sup.5 .about.646 5.9 C,D 3.2 .times. 10.sup.5 .about.492 6.5 E,F 2.3 .times. 10.sup.5 .about.354 6.7 G 2.1 .times. 10.sup.5 .about.323 7.0 H 1.2 .times. 10.sup.5 .about.185 7.2 I 1.0 .times. 10.sup.5 .about.154 7.3 J 8.7 .times. 10.sup.4 .about.134 7.4 K 7.4 .times. 10.sup.4 .about.114 7.5 ______________________________________
The Jeppesen work demonstrates two important points: (1) that in large gel formats the further separation of DNA fragments larger than their pore limit size is not observed; and (2) that polyacrylamide gel concentrations (%T/%C) only up to certain identified levels are useful to separate a given range of DNA fragments by the "normal gel sieving process".
Nucleic acid fragment analysis using denaturing polyacrylamide gels was described at least by Maniatis et al., Biochemistry, 14:387 (1975). Maniatis (1975) described the relative electrophoretic mobilities of RNA and DNA molecules having chain lengths of 10-150 nucleotides (nt) when separated in 12% polyacrylamide/3.3% crosslinker (N,N'-methylene bisacrylamide) (12%T/3.3%C) gels containing 7M urea (denaturing gel), using gel dimensions of 20cm .times.20 cm.times.0.15 cm and running conditions of 1.times. TBE buffer for several hours at a constant voltage of 10 volts per centimeter (10V/cm) in the vertical direction. Sanger et al., Nature, 265:687 (1977), showed the relative mobility of PhiX174 DNA/Hind II fragments with chain lengths of 1,049, 770, 609, 495, 393, 335/340/345 (unresolved triplet), 297/291 (unresolved doublet), 163, and 79 nt in 5% polyacrylamide gel containing 98% formamide. Sanger's gel dimensions were 20cm .times.20 cm and the gels were run in 0.02M phosphate buffer. Maniatis et al., in "Methods in Enzymology", vol. 65, part 1, eds. Grossman et al, Academic Press, N.Y., p 299 (1980), recommends the above general conditions for separation of short to intermediate size DNA and RNA fragments under denaturing conditions.
Sambrook et al., in "Molecular Cloning: A Laboratory Manual," 2nd edition, Cold Spring Harbor, N.Y., pp. 6.2 to 6.63 (1989), and the references contained within, recommend the following gel concentrations and conditions for separating nucleic acid fragments on agarose gels having fragment sizes in the range of 100 to 60,000 nt at the following percentages:
TABLE 2 ______________________________________ Range of Sizes for Agarose in Gel (%) DNA Molecules (nt) ______________________________________ 0.3 5,000-60,000 0.6 1,000-20,000 0.7 800-10,000 0.9 500-7,000 1.2 400-6,000 1.5 200-3,000 2.0 100-2,000 ______________________________________
Following the above recommendations, agarose gel electrophoresis of DNA was generally carried out in the horizontal direction using slab gels ranging from 14 to 20 centimeters in length. It was generally recommended that the gels be run at no more than 5 V/cm. Depending upon the degree of resolution required and the voltage utilized, running times for agarose gels varied from one to sixteen hours. However, agarose gels have very poor resolution below 100 nt, and only intermediate resolution at higher chain lengths (&gt;100 nt). Therefore, agarose gels are more frequently used for Southern analysis or restriction fragment analysis, rather than for DNA sequencing applications which requires high resolution of shorter chain lengths.
Very large linear ds-DNA molecules were found to migrate through agarose gels at the same rate. The limit of resolution was reached when the radius of gyration of the linear DNA duplex exceeds the pore size of the gel. At that point the DNA can no longer be sieved by the gel according to size but must now migrate end-on through the narrow pores. This process of end-on migration is known as "reptation". Sambrook et al., in "Molecular Cloning: A Laboratory Manual" 2nd edition, Cold Spring Harbor, N.Y., pp. 6.2 to 6.63 (1989). One solution to the problem of separating large DNA molecules is a technique called pulsed field electrophoresis developed at least by Schwartz et al., Cell, 37:67 (1984). In this method, pulsed, alternating, orthogonal electric fields are applied to agarose gels. The large DNA molecules become trapped in their "reptation tubes" and can make no further progress through the gel until they have reoriented along the new axis of the electric field. The larger DNA molecules require a longer reorientation time; the smaller molecules with reorientation times less than the pulse begin to separate according to size. Pulse field electrophoresis involves large agarose gel formats, complex electrode arrangements, and very long running times to achieve separations. Pulse field electrophoresis provides important background information for the present invention because it shows; first, that very large DNA molecules which are larger than the gel pore size are not separated using regular large scale gel formats and procedures; and second, that the solution to the "reptation problem" is the complicated and extremely long process described above.
Sambrook et al., in "Molecular Cloning: A Laboratory Manual", 2nd edition, Cold Spring Harbor, N.Y., pp. 6.2 to 6.63 (1989), and the references contained within, recommend the following gel concentrations and conditions for separating nucleic acid fragments on non-denaturing polyacrylamide gels in the following concentration ranges where the DNA fragments have sizes in the range of 6 to 2000 nt in length:
TABLE 3 ______________________________________ polyacrylamide Range of Sizes (% T/% C) for DNA Molecules (nt) ______________________________________ 3.5% T/3.3% C 100-2000 5.0% T/3.3% C 80-500 8.0% T/3.3% C 60-400 12.0% T/3.3% C 40-200 15.0% T/3.3% C 25-150 20.0% T/3.3% C 6-100 ______________________________________
Following the above recommendations, gels were typically run in vertical format and over lengths from 10 to 100 centimeters depending on the resolution required. Gels were run in 1.times. TBE buffer at voltage gradients between 1 V/cm to 8 V/cm. Higher voltages were not recommended due to the problems associated with overheating. Runs generally took from one to twelve hours depending upon resolution required. Strand-separating polyacrylamide gels were recommended for nucleic acid fragments below 1000 nt in length, in particular for sequencing by the Maxam-Gilbert procedure [Maxam et al., Proc. Natl. Acad. Sci. U. S. A., 74:560 (1977)] and for hybridization to low abundance RNA's (Maniatis et al., in "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor, N.Y., pp. 179-185, 1982). For DNA fragments greater than 200 nt in length, 5%T/2%C gels were recommended; while 8%T/3%C gels were recommended for fragments less than 200 nt in length. These gels were typically run in 1.times. TBE and at 8 V/cm.
Sambrook et al., in "Molecular Cloning: A Laboratory Manual", 2nd edition, Cold Spring Harbor, N.Y., pp. 6.2 to 6.63 (1989), and the references contained within, recommend the following gel concentrations and conditions for separating nucleic acid fragments on denaturing polyacrylamide gels containing 7M urea, where the duplex DNA fragments are in the 10 to &gt;200 nt range:
TABLE 4 ______________________________________ polyacrylamide Range of Sizes for (% T/% C) DNA Molecules (nt) ______________________________________ 4% T/5% C &gt;200 5% T/5% C 80-200 8% T/5% C 40-100 12% T/5% C 10-50 ______________________________________
Following the above recommendations, denaturing gels were typically run at 20 V/cm in 1.times. TBE, and it was typically recommended that the DNA fragments be allowed to migrate the full length of the gel in order to obtain maximum separation. It should be pointed out that the agarose and polyacrylamide gel formulations discussed above have been frequently used in so-called mini-gel formats, which are generally about five to ten centimeters in length. Mini-gels represent a two to four fold size reduction in the linear dimension, and are basically only scaled down versions of the systems described above. As will be discussed below, the high resolution necessary for DNA sequencing is not achieved when accepted large-scale procedures are scaled down to mini-gel format. More importantly, as will be demonstrated by the present disclosure, true microelectrophoresis which produces the required resolution can not be obtained by simply scaling down large scale procedures.
In the case of DNA sequencing, where separation of DNA or RNA fragments differing by a single nucleotide is required, electrophoresis techniques with speed and high resolution are required. Maxam et al developed the chemical cleavage sequencing method [Proc. Natl. Acad. Sci. U. S. A., 74:560 (1977)] and used the following polyacrylamide compositions and procedures for sequencing gels: for DNA sequences from 1 to 30 nt a 20%T/5%C (8.3M Urea) gel was used; for sequences from 25 to 250 nt a 8%T/5%C (8.3M Urea) gel was used; and for sequences greater than 250 nt a 6%T/5%C gel was used. The gels were 20 cm.times.40 cm in width and length, respectively, and gel thicknesses of 1.5 mm, 0.5 mm, and 0.3 mm were used. The thinner gels of 0.3 mm were typically preferred due to their capacity to withstand higher voltages and faster run times. Sequencing gels were run in 1.times. TBE buffer at voltages of 25 V/cm for 1.5 mm gels, and 50 V/cm for the 0.3 mm gels. See, for example, Maxam et al., in "Methods in Enzymology", vol. 65, part 1, eds., Academic Press, N.Y., p. 499 (1980). Depending on the separation required, resolution of the DNA fragments in the sequencing reactions takes many hours to complete.
More recently Barker described in "Nucleic Acid Sequencing: A Practical Approach", eds. et al, IRL Press, N.Y., Chapter 5, 117 (1989), the use of one meter long gels for DNA sequencing by the Maxam and Gilbert method. Early workers carrying out the primed synthesis DNA sequencing method [Sanger et al., Proc. Natl. Acad. Sci. U. S. A., 74:5463 (1977)] used the following polyacrylamide compositions and procedures for sequencing gels: for DNA fragments from 30 to 250 nt an 8%T/5%C (7M urea) gel was used, and for fragments &gt;250 nt a 6%T/5%C (7M urea) gel was used. See, for example, Smith in "Methods in Enzymology", vol. 65, part 1, eds. Grossman et al., Academic Press, N.Y., p. 560 (1980); and Sanger et al, FEBS Lett., 87:107 (1978). The gels were 20 cm.times.40 cm, and 0.35 mm in thickness. Gels were run at 30 to 40 volts/cm and take many hours to complete. In general, as DNA sequencing developed, the trend in gel formulations and size of polyacrylamide slab gels progresses toward thinner gels and slightly less concentrated gels, but there were no significant reductions in the length of these sequencing gels. See, for example, Andrews in "Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications", Oxford University Press, N.Y., chapter 6, pp. 148-177 (1986); and "Nucleic Acid Sequencing: A Practical Approach", eds. Howe et al, IRL Press, N.Y., (1989), and references contained within.
Automated DNA sequence analyzers based on fluorescent detection have become available in the past few years. Workers at Applied Biosystems, Inc. (ABI) and the California Institute of Technology have developed an automated DNA sequencer which uses four fluorescent dye-labelled primers. Connel et al. BioTechniques, 5:342 (1987). Each of the dye-labelled primers is paired with one of the four dideoxynucleoside triphosphate chain terminators, and used in the Sanger sequencing method to introduce fluorescent labels into DNA fragments produced by primer extension. More recently, fluorescent dideoxynucleotide nucleotide derivatives have become available for use on the ABI sequencing machines, thus eliminating the need for fluorescent primers. The fluorescent fragments produced in the separate A, C, G, and T reactions are combined and can be co-electrophoresed in the same lane and distinguished during electrophoresis by the color of their fluorescence. The system has an argon-ion laser which excites each of the fluorescent fragments as they pass through a small area near the bottom of the separation gel. The fluorescent signal from the fragments is focused by a collection lens through a four wavelength selectable filter onto a photomultiplier tube (PMT). The digitalized signal from the PMT is transferred directly into a computer for subsequent processing and display.
After computer processing, the sequence information is presented graphically as a linear array of colored peaks with the actual base sequence (A, T, G, & C) given above each peak. The system uses 6%T/5%C (7M Urea) polyacrylamide gels, 0.4 mm or less in thickness. An 8% gel is recommended for better resolution closer to the primer; while a 4% gel can be used for better downstream resolution (&gt;400 bp). A running distance of 25 cm is required to achieve the appropriate resolution of the fluorescent DNA fragments. Generally, electrophoresis is carried out at 30 to 40 volts/cm, with sequences up to 500 bases being determined in a 8 to 12 hour run. Heiner et al, in "Nucleic Acid Sequencing: A Practical Approach" eds. Howe et al, IRL Press, N.Y., Chapter 8, pp.221-235 (1989).
Another automated DNA sequencer using fluorescent detection has been developed at the E. I. du Pont Company as described by Prober et al. Science, 238:336 (1987). This system uses an 8%T/5%C (7M Urea) gel in a 20 cm wide by 40 cm long by 0.3 mm thick format. Thus, the state of the art for the high resolution separation of DNA fragments on polyacrylamide slab gels for sequencing purposes: (1) uses gel concentrations of about 6%T/5%C, (2) requires gel lengths of at least 25 to 40 cm, (3) is limited to voltages of 40 volts/cm, and (4) requires many hours of running time.
A newer technique, which may be considered second generation DNA sequencing technology, is capillary gel electrophoresis. Morris et al, U.S. Pat. No. 4,909,919; Luckey et al., Nucl. Acids Res., 18:4417 (1990); Drossman, Anal. Chem., 62:900 (1990); and Guttman et al., Anal. Chem., 62:137 (1990). Capillary electrophoresis involves the use of very fine glass capillary tubes 50 to 100 .mu.m in diameter and 40 cm to 100 cm in length. Capillary gel electrophoresis has an advantage in that much greater electric fields may be applied, because of the reduced Joule heating in the small diameter capillary. This results in as much as a 14 fold faster separation speed over conventional slab gel methodologies. Thus, whereas a DNA sequencing separation of 300-400 bases run at 30 to 40 volts/cm on a 40 cm slab gel takes 7-8 hours to complete, the same separation on a 40 cm capillary gel run at over 400 volts/cm takes only 30-40 minutes to complete. The lower concentration gel [3.2%T/2.7%C (7M Urea)] used in the capillary columns is one factor in the improved separation speed.
Thus while capillary gel electrophoresis represents a significant improvement in separation speed, the gel lengths necessary for achieving the separation are still 40 to 70 cm long, and very low polyacrylamide concentrations are utilized.